The proposed research is a mechanistic study of the reaction of thrombin and other proteases with the plasma coagulation inhibitors Alpha2-macroglobulin (Alpha2-M) and antithrombin (AT). The long-range goal is to describe the sequence of reactions in enzyme inhibition in the circulation: results bear on the role of these proteins in blood coagulation and defense against exogenous proteases. The understanding of mechanism provides a theoretical framework for clinical assay, therapy and design of drugs. For Alpha2M, we intend to examine the various aspects of the reaction with protease--the formation of covalent and non-covalent complexes, specific proteolysis and reactivity with methylamine. The kinetics of these changes will be followed in parallel in order to understand causal relations between them. Reactions will be studied with native enzymes, Gamma-glutamyl-Epsilon-lysyl bond involving enzyme amino groups and the glutamyl residue of the methylamine site of Alpha2M. In the AT work, we propose to develop fluorescence methods for kinetic studies of the reaction with proteases under a wide range of conditions of temperatures, pH and reactant concentrations. We want to understand the role of binding of heparin, particularly in the ternary thrombin-AT-heparin complex. The techniques used will be measurement of instrinsic fluorescent changes currently studied in this laboratory, polarization of fluorescence using an active dansyl-thrombin we have prepared, and radiationless energy transfer using labelled heparin. We will study Alpha and Gamma-thrombin and other proteases as well as two derivatives of AT: 1) a form prepared by mild reduction which still has thrombin-inhibiting ability but does not bind heparin, and 2) a modified AT which is not an enzyme inhibitor but binds to heparin-agarose.